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. 2016;16(1-2):95-110.
doi: 10.1159/000440864.

AF710B, a Novel M1/σ1 Agonist With Therapeutic Efficacy in Animal Models of Alzheimer’s Disease

Free PMC article

AF710B, a Novel M1/σ1 Agonist With Therapeutic Efficacy in Animal Models of Alzheimer’s Disease

Abraham Fisher et al. Neurodegener Dis. .
Free PMC article

Abstract

We previously developed orthosteric M1 muscarinic agonists (e.g. AF102B, AF267B and AF292), which act as cognitive enhancers and potential disease modifiers. We now report on a novel compound, AF710B, a highly potent and selective allosteric M1 muscarinic and σ1 receptor agonist. AF710B exhibits an allosteric agonistic profile on the M1 muscarinic receptor; very low concentrations of AF710B significantly potentiated the binding and efficacy of carbachol on M1 receptors and their downstream effects (p-ERK1/2, p-CREB). AF710B (1-30 µg/kg, p.o.) was a potent and safe cognitive enhancer in rats treated with the M1 antagonist trihexyphenidyl (passive avoidance impairment). These effects of AF710B involve σ1 receptor activation. In agreement with its antiamnesic properties, AF710B (at 30 nM), via activation of M1 and a possible involvement of σ1 receptors, rescued mushroom synapse loss in PS1-KI and APP-KI neuronal cultures, while AF267B (1 µM) was less potent in PS1-KI and ineffective in APP-KI models, respectively. In female 3xTg-AD mice, AF710B (10 µg/kg, i.p./daily/2 months) (i) mitigated cognitive impairments in the Morris water maze; (ii) decreased BACE1, GSK3β activity, p25/CDK5, neuroinflammation, soluble and insoluble Aβ40, Aβ42, plaques and tau pathologies. AF710B differs from conventional σ1 and M1 muscarinic (orthosteric, allosteric or bitopic) agonists. These results highlight AF710B as a potential treatment for Alzheimer's disease (e.g. improving cognitive deficits, synaptic loss, amyloid and tau pathologies, and neuroinflammation) with a superior profile over a plethora of other therapeutic strategies.

Figures

Figure 1
Figure 1. AF710B is a selective ligand for M1 mAChR and σ1R, potentiates the effects induced by carbachol and improves cognition in a passive avoidance test in trihexyphenidyl-treated rats
(A) In high-throughput receptogram profiling AF710B emerged as a highly selective ligand for M1 mAChR and σ1R, respectively as shown by displacement of tritiated-pirenzepine (an M1 mAChR radioligand) from rat cerebral cortex and tritiated-pentazocine (a σ1R radioligand) from guinea-pig cerebral cortex. (B) Very low concetrations of AF710B potentiates both carbachol-induced displacement of tritiated-pirenzepine from rat cerebral cortex and the efficacy of carbachol as evidenced by the ratio of KL/KH. (C) Very low concentrations of AF710B potentiated carbachol-induced phospho-Extracellular-signal-regulated kinases (pERK1/2) in starved proliferated PC12M1 cells. *p<0.001 vs. carbachol, 10 nM. (D) Very low concentrations of AF710B potentiated carbachol-induced phospho-CREB (cAMP response element-binding) in starved proliferated PC12M1 cells. **p< 0.001, *p<0.01 vs. carbachol, 10nM. (E) AF710B reverts the cognitive decline induced by trihexyphenidyl, an M1 mAChR antagonist. In Experiments 1 and 2 the retention latencies of trihexyphenidyl -treated rats with double distilled water (DDW) were significantly shorter than that of control rats treated with DDW (p<0.001). Experiment 1 and 2 were combined as no significant differences were observed between the trihexyphenidyl-treated rats with DDW in both experiments. The retention latency of trihexyphenidyl- treated rats with AF710B (1, 3, 10 µg/kg, po) and AF710B (30 µg/kg, po) were significantly longer than that of trihexyphenidyl-treated rats with DDW. The retention latency of trihexyphenidyl-treated rats with AF710B 100 µg/kg, po was not different from that of trihexyphenidyl-treated rats with DDW. *p<0.01, **p<0.001, compared to trihexyphenidyl + DDW.#p<0.01, compared to trihexyphenidyl + 100 µg/kg, po of AF710B. (F) Retention latency of trihexyphenidyl-treated rats with DDW was significantly shorter than that of control rats treated with (p<0.001). The retention latency of trihexyphenidyl-treated rats with AF710B 10µg/kg was significantly longer and the σ1R antagonist NE 100 blocks the effects of AF710B. # p< 0.02, vs Saline + AF710B + Trihexyphenidyl; * p<0.05, vs. Saline + AF710B + Saline; ** p<0.01, vs. Saline + AF710B + Trihexyphenidyl; *** p< 0.001, vs. Saline+ DDW+ Saline; ***p< 0.001, vs. NE-100 + AF710B + Saline.
Figure 2
Figure 2. Activation of M1 mAChR/σ1R complex by AF710B restores synaptic spines
(A–B) Primary hippocampal neuron cultures were prepared from wild type non-transgenic (nTg), PS1-M146V-KI (M146V.KI) and APP-KI (APP.KI) pups. Hippocampal neurons were transfected with TdTomato to visualize dendritic spine morphology. By DIV16 the prevalence of mushroom spines decreased in both AD models relative to wild type cultures. 16-hour treatment of cultures with 30 nM AF710B prevented the loss of mushroom spines, whereas AF267B (1µM) was only partially effective in M146V.KI cultures. (C–D) In APP.KI cultures, the spine rescue by AF710B was unaffected by the σ1R antagonist NE100 (1 µM). Pirenzepine (1 µM) completely blocked the spine rescue by AF710B. The addition of NE100 had no additional effect. (E–G) Wild type hippocampal neuron cultures were transfected by lenti-viruses on DIV8 with plasmids encoding siRNA against σ1R (or scrambled siRNA as a control). Cells were harvested for western blotting on DIV17 to verify σ1R knockdown (F). (E,G) σ1R knockdown reduced the prevalence of mushroom spines to similar levels as in AD models. AF710B was unable to rescue mushroom spine loss from σ1R knockdown. *p<0.05, **p<0.001 and ***p<0.0001 in comparison to the respective control condition. ##p<0.001 in comparison to AF710B.
Figure 3
Figure 3. AF710B reduces AD-like pathology in the 3xTg-AD mouse model
(A,B) AF710B (10 µg/kg/day, ip) improves cognition in 12-month old 3xTgAD mice, as demonstrated by the (A) decrease in escape latency times to the platform, but not on the 6th day and (B) increase in the amount of time in the target quadrant compared to vehicle-treated 3xTgAD during the probe trial, in the Morris water maze. **P<0.01 between vehicle-treated nTg and 3xTgAD mice, #p< 0.05 between vehicle- and AF710B-treated 3xTgAD mice. (C) ELISA analysis reveals that levels of soluble and insoluble Aβ40 and Aβ42 are decreased in in 3xTgAD treated with AF710B (10 µg/kg/day). (D) Thioflavin S-positive plaques are also reduced in AF710B-treated 3xTgAD mice, as shown by staining and volumetric analysis. (E) BACE1 and the C-terminal fragment of BACE cleavage of APP, C99, are significantly decreased in AF710B-treated 3xTgAD mice, while full-length APP and non-amyloidogenic enzymes ADAM10 and ADAM17 remain unchanged. Quantification of western blots was performed by densiometric analysis and is presented as a percentage of control, normalized to GAPDH. Activation of M1R/σ1R complex also reduces the phosphorylation of tau in the (F) AT-100 and (G) AT-8 epitopes in the 3xTg-AD mouse model. AF710B treatment results in the reduction of several phosphoepitopes of tau, while total tau levels remain the same in versus vehicle-treated 3xTgAD mice. (H) The reduction in tau phosphorylation seen in AF710B-treated 3xTgAD mice is mediated through GSK3β and CDK5, as demonstrated by the higher phosphorylation at Ser9 of GSK3β and reduced levels of the CDK5 activator peptide p25, respectively. Quantification of western blots was performed by densiometric analysis and is presented as a percentage of control, normalized to GAPDH. (I) AF710B reduces inflammation, as we found significant reduction in the number of GFAP+-astrocytes and Iba-1+-microglia in the vicinity of 6E10+-plaques. Graphs represent the volumetric analysis of Aβ, astrocytes and microglia. *P<0.05 and **P<0.01 between vehicle- and AF710B-treated 3xTgAD mice.

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